During the next period of support we, will continue our studies on the coordinate regulation of virulence gene expression in three different bacterial species, Salmonella typhimurium, Vibrio cholerae and Bordetella pertussis. In order to facilitate the identification of virulence genes, we have devised a genetic strategy that allows the direct selection of genes that are expressed in animal tissues. We call this methodology "in vivo expression technology (IVET)" and now propose to identify genes in each of the three target organisms that show a pattern of regulation that is consistent with a virulence gene (i.e., expressed poorly during grow on laboratory media but highly expressed in animal tissues). The gene fusions displaying this regulation will be cloned and the nucleotide sequence of the fusion junction determined. The wild-type target gene will also be cloned and used to construct corresponding null mutations in the target organism. These mutants will be tested for virulence defects in appropriate animal models. The particular regulatory response of selected fusions will be studied in vitro in an effort to ascertain the host environmental signals controlling virulence gene expression. The regulatory genes mediating the host specific regulatory responses will be identified by genetic methods (e.g., isolation of constitutive mutations that derepress the fusion in vitro). We will also isolate new gene fusions that are coordinately regulated by the same regulatory system. We further propose to construct a series of improved IVET vectors one of which will allow identification of genes encoding secreted or membrane proteins that are expressed selectively in host tissues, while another that will provide a sensitive measurement of tissue-specific gene expression by using site-specific recombination as a reporter. Ongoing studies that will also be continued will include studies in S. typhimurium that will address the regulation of O-antigen synthesis by an in vivo induced anti-sense RNA. In B. pertussis, we will study the induction of Vir-repressed genes by intracellular growth. Finally, in V. cholerae we will study the temporal phasing of gene expression during the infection cycle focusing on genes that are both activated as well as repressed by the ToxR regulatory system and the heat shock response. These experiments should provide several practical applications in vaccine and antimicrobial drug development.